Superovulation of Holstein heifers by a single subcutaneous injection of FSH dissolved in polyvinylpyrrolidone

This study was undertaken to determine whether a single injection of porcine FSH (pFSH) would induce a superovulatory response in cattle. Holstein heifers were given a single injection of pFSH (30 mg, s.c.) dissolved in saline (Group 1, n = 5); 50% polyvinylpyrrolidone (PVP; Group 2, n = 5); or 25% PVP (Group 3, n = 4). Group-4 heifers (n = 5) were given multiple intramuscular injections of pFSH every 12 h for 3 d at decreasing doses, for a total of 30 mg. All animals received a single injection of 750 microg PGF2 alpha 48 h after the initiation of pFSH treatment. Animals exhibiting estrus were artificially inseminated twice throughout estrus. Ova and embryos were recovered nonsurgically. Ovaries were examined by transrectal ultrasonography or by palpation per rectum on Day 7 or 8 of estrus. Plasma concentrations of pFSH, bovine FSH progesterone, estradiol-17 beta and inhibin were determined by specific radioimmunoassays. The number of corpora lutea (CL) and the numbers of total and transferable embryos which were detected and recovered in Groups 2 and 3 were equivalent to the numbers detected and recovered in Group 4. In Group 1, however, only 1 of 5 animals ovulated even a single oocyte. The present study demonstrated that only a single injection of pFSH dissolved in PVP was capable of inducing a superovulatory response by maintaining a high plasma FSH concentration to allow for the recovery of a sufficient number of embryos for transplantation.     

Superovulation of a low fecundity sheep breed using a porcine gonadotrophin extract with a defined LH content (Pluset)

The aim of this study was to determine the efficiency of a porcine pituitary gonadotrophin extract with a defined pLH content in the superovulation of sheep. Estrus was synchronized in 61 Polish Mountain ewes with intravaginal fluorogestone acetate sponges. Twenty-four hours before the sponges were removed, the ewes underwent different superovulatory treatments: Group I 250 IU of pFSH with 250 IU of pLH (n=19); Group II 500 IU of pFSH with 500 IU of pLH (n=19); and Group III 750 IU of pFSH and 750 IU of pLH (n=18). Gonadotrophine was administered intramuscularly twice a day over a 3-day period in decreasing dosages. A control group of ewes (n=5) was treated with saline. In most of the ewes estrus began about 20 hours after sponges were removed. All the ewes were bred naturally every 12 hours. Superovulation was confirmed in 75% of the treated animals. The ewes receiving 250 IU each of pFSH and pLH produced an average of 7.6 +/- 3.1 corpora lutea (CL), 6.3 +/- 2.4 ova and 4.3 +/- 4.1 transferable embryos. Group II (500 IU of pFSH and pLH) produced 8.5 +/- 4.0 CL, 7.6 +/- 4.1 ova, and 4.1 +/- 2.9 transferable embryos. Group III (750 IU each of pFSH and pLH) produced 8.3 +/- 5.2 CL, 7.5 +/- 5.5 ova and 5.2 +/- 5.1 transferable embryos. The mean embryo recovery rate was 87% for all three groups. Differences in superovulatory response and embryo recovery rate among the groups were not statistically significant (P>0.05).     

Fertility and blood progesterone levels following LHRH-induced superovulation in FSH-treated anestrous goats

Twenty mature, mixed-breed, seasonally anestrous female goats were used to study the effects of luteinizing hormone releasing hormone (LHRH) on ovulation rate, fertility, and blood progesterone levels following norgestomet-induced estrus and follicle stimulating hormone (FSH) treatments. Each goat received 6 mg norgestomet by subcutaneous (sc) implant and 3 mg intramuscularly, along with an intramuscular (im) injection of 5 mg estradiol valerate. Four injections of FSH were given for 2 d in divided doses of 10, 10, 5 and 5 mg im every 12 h, starting at 24 h before implant removal. The goats were randomly assigned to 1 of 2 equal treatment groups, and were treated with 2 intravenous (iv) injections of either 0.9% saline (control) or 300 ug LHRH at 24 and 48 h after the removal of the implants. All the goats exhibited estrus within 24 or 36 h of implant withdrawal and were mated to bucks of proven fertility. At laparotomy on Day 7 or 8 after the removal of the implants, the mean number of unovulated follicles was higher (P<0.05) in Group I than in Group II. The mean number of corpora lutea (ovulation rate), the total number of embryos and the number of normal embryos recovered were higher (P<0.05) in LHRH-treated does than in the controls. Treatment with LHRH resulted in 72.14% fertility (mean number of CL = 14) as compared with the controls with 64.29% fertility (mean number of CL = 2.8). The embryos obtained from goats in Group II were of more uniform developmental age regardless of the day of embryo collection, as compared with those of the controls. Plasma progesterone levels were significantly increased on Days 4 to 6 in both treatment groups. The results of this study have demonstrated that the FSH and LHRH treatment regimen increased follicular development, ovulation rate and blood progesterone levels in norgestomet-treated anestrous goats. Moreover, LHRH treatment enhanced fertility, and improved embryo quality as indicated by the significantly higher total number of embryos as well as the higher (P<0.05) number of normal recoverable embryos.     

Effect of timing of prostaglandin PGF 2 alpha injection subsequent to embryo collection on the resumption of normal follicular development following superovulatory treatment in cattle

Nonlactating Holstein and Jersey cows (n = 24) were superovulated and ovarian follicular development was monitored by transrectal ultrasound during the period after embryo recovery. Luteolysis was induced by two injections of prostaglandin F(2)alpha (PGF; 25 mg Lutalyse; 12-h interval) at specific times after superovulatory induced estrus (Treatment 1, Day 9; Treatment 2, Day 12; Treatment 3, Day 17; Treatment 4, Day 25; superovulatory estrus = Day 0 of Cycle 1). Follicular development was monitored during Cycle 1 before and after PGF injection and continued through the ensuing estrous cycle (Cycle 2). Superovulation led to more than one embryo collected in 14 cows (mean = 8.71 embryos: positive superovulatory response [PSR] cows), while 10 cows were not successfully superovulated (mean = 0.1 embryo; negative superovulatory response [NSR] cows). These cows differed in terms of number of unovulated follicles detected at embryo collection (4.21 vs 17.2, PSR vs NSR) and plasma progesterone during the superovulatory estrous cycle (32.3 ng/ml PSR vs 8.6 ng/ml NSR). Follicular development during Cycle 1 started sooner in NSR than in PSR cows (day by class by response P<0.03) and was initiated on Days 11 to 12 in NSR cows and on Days 19 to 20 in PSR cows. Interval to estrus after PGF averaged 6.3 d. Cows having short intervals to estrus had follicles at the time of PGF injection. Treatment influenced the length of Cycle 1, but it did not affect the interval to estrus after PGF, the length of Cycle 2, or follicular development during Cycle 2. The results indicate that 1) the timing of PGF injection after embryo collection does not influence subsequent follicular populations, 2) elongated estrous cycles and intervals to estrus after PGF in superovulated cattle are a function of decreased follicular activity, and 3) the presence of numerous corpora lutea and not the superovulatory treatment, per se, seem to attenuate follicular growth.     

Superovulatory response to a single subcutaneous injection of Folltropin-V in beef cattle

A series of 4 experiments were designed to evaluate the feasibility of superstimulation in beef cattle with a single sc injection of the porcine pituitary extract, Folltropin-V. In the preliminary study (Experiment 1), superovulatory response of cows (n=7) treated with a single sc injection of 400 mg NIH-FSH-P1 Folltropin-V was not different than that of cows (n=8) superstimulated with twice daily im injections over 4 d, or a single sc injection plus an injection of eCG (n=12). Experiments 2 and 3 were designed to determine the optimal site of a single sc injection. In Experiment 2, cows (n=25) with body condition scores (BCS) of 1 to 2 were used. The mean number of CL counted and ova/embryos collected was lower (P<0.05) in cows treated with the single sc injection in the neck region than in cows treated with a single sc injection behind the shoulder, or with the twice daily im injection treatment. In Experiment 3, cows (n=49) with BCS of 3 to 5 were used. There were no differences in the number of CL, total ova/embrvos collected, fertilized ova and transferable embryos whether treatments were given in the neck region or behind the shoulder, or whether the cows were implanted or not implanted with Syncro-Mate-B. Experiment 4 was designed to determine the optimal superstimulatory dosage of Folltropin-V administered by a single sc injection. Superovulatory response of cows treated with the higher doses (400 mg, 600 mg or 800 mg NIH-FSH-P1) was higher (P<0.05) than those treated with 200 mg NIH-FSH-P1. The number of unovulated (>/=10 mm) follicles at the time of ova/embryo collection was higher (P<0.05) in the 600 and 800 mg groups, and progesterone concentration at estrus was higher (P<0.05) in cows treated with 800 mg than with 400 or 200 mg. It was concluded that a single, bolus sc injection of 400 mg NIH-FSH-P1 of Folltropin-V is as efficacious as the 4-d, twice daily im treatment protocol for inducing superovulation in beef cows. The amount of subcutaneous fat and site of injection appeared to affect the efficacy of a single sc injection; a single bolus sc injection of Folltropin-V behind the shoulder resulted in the most predictable superovulatory response.     

Superovulatory response of Sistani cattle to three different doses of FSH during winter and summer

Objective of the present study was to investigate the effect of season and dose of FSH on superovulatory responses in Iranian Bos indicus beef cattle (Sistani). Cyclic cows, in summer (n=16) and winter (n=16), were assigned randomly to three dose-treatment groups of 120 (n=10), 160 (n=12) and 200 (n=10) total mg of Folltropin-V with injections given twice daily for 4 days in decreasing doses. Estrous cycles were synchronized with two prostaglandin F2alpha injections given 14 days apart. From day 5 after the ensuing cycle, daily ovarian ultrasonography was conducted to determine emergence of the second follicular wave at which time superovulation was initiated. Relative humidity, environmental and rectal temperatures were measured at 08:00, 14:00 and 20:00 h for the 3 days before and 2 days after the estrus of superovulation. Non-surgical embryo recovery was performed on day 7 after estrus. The effects of season, dose, time of estrous expression and all two-way interactions were evaluated on superovulatory responses: total numbers of CL, unovulated follicles (10 mm), ova/embryo, transferable and non-transferable embryos. Season (summer or winter), doses of Folltropin-V (120, 160 or 200 mg NIH) and time of estrous expression (08:00, 14:00 or 20:00 h) did not affect the number of transferable embryos (3.1+/-0.58). When superovulatory estrus was detected at 08:00, a FSH dose effect was detected with the greatest numbers of CL (12.2+/-0.87) and total ova/embryos (12.2+/-1.46) occurring with 200 mg FSH (dosextime of estrous expression; P<0.01).     

Ultrasonic study of follicular dynamics following superovulation in German Merino ewes

Ewes are commonly superovulated with a single dose of eCG or multiple doses of pFSH. It would be convenient and less expensive to use a single dose of FSH, but results of various trials have been controversial. We wished to investigate ovarian dynamics using ultrasonography after superovulation with a single dose of pFSH and hMG as compared with a single dose of eCG. Estrus was synchronized during the breeding season with fluorogestone acetate-containing intravaginal sponges in adult German Merino ewes (n = 38). They were superovulated with single doses of pFSH (17 mg; n = 10), hMG (600 IU FSH and 600 IU LH; n = 9) or eCG (1250 IU; n = 10) given at the time of sponge removal, or pFSH (17 mg; n = 9) given 36h before sponge removal. Follicular and luteal development were observed by ultrasonic scanning every 8 h from the gonadotrophin injection until the end of estrus, and then once daily until Day 6 after estrus. Jugular venous blood was collected starting immediately before and 1 h after superovulation treatment, then twice daily until the end of estrus and once daily for the following 7 days. Concentrations of estradiol-17beta (E2) and progesterone (P4) were measured in plasma. Differences in the follicular dynamics of the 4 superovulation groups were obvious. The functional duration of the pFSH action was estimated to last approximately 48 h, whereas eCG and hMG were active for up to 72 h. The diameter of the ovulatory follicles proved to be smaller than it was described for unstimulated ewes. Single applications of pFSH or hMG can induce a superovulatory response, although the post-estrus progesterone profile revealed a high premature luteal regression rate in the different superovulation groups. Premature corpus luteum regression could not be seen by ultrasonography at this early stage of the luteal phase, indicating that the technique may fail to detect these corpora lutea in an embryo transfer program. However, ultrasonography represents a suitable method to observe follicular dynamics following different superovulation regimens in sheep.     

Superovulatory response following transvaginal follicle ablation in cattle

A study was designed to compare superovulatory responses in cattle when gonadotropin treatment followed 1 of 3 different treatments to synchronize follicular wave emergence. Animals at unknown stages of the estrous cycle were randomly assigned to 3 groups: ablation of the 2 largest follicles per pair of ovaries (n = 21); ablation of all follicles > or = 5 mm (n = 19); or intramuscular administration of 5 mg estradiol-17beta plus 100 mg progesterone (n = 23). All animals were given a CIDR-B intravaginally at the time of the respective treatments. Gonadotropin treatment, initiated 1 d after follicle ablation or 4 d after estradiol plus progesterone treatment, in the respective groups, consisted of 200 mg of pFSH divided in decreasing doses twice daily over 4 d. Cloprostenol (500 microg) was given at 48 and 60 h after the first pFSH treatment; CIDR-B devices were removed at the time of the second cloprostenol treatment. Ovarian ultrasonography was done on the days of CIDR-B insertion, first gonadotropin treatment, and at 36 and 72 h after CIDR-B removal. Cattle were inseminated twice, at 60 and 72 h after the first injection of cloprostenol. Ovarian and ova/embryo data were collected at slaughter 5, 6 or 7 d after insemination. No differences were detected among groups in the number of follicles > or = 8 mm at the time of first insemination (20.4 +/- 1.7 vs 16.6 +/- 2.0 vs 19.9 +/- 2.3; P > 0.05). At slaughter, no differences were detected among groups in the numbers of CL (23.3 +/- 1.9 vs 17.9 +/- 1.9 vs 20.1 +/- 2.6; P < 0.05), unovulated follicles > or = 8 mm (2.2 +/- 0.5 vs 2.1 +/- 0.3 vs 3.7 +/- 0.9; P < 0.05), ova/embryos (11.0 +/- 1.4 vs 12.2 +/- 1.3 vs 8.5 +/- 1.3; P < 0.05), fertilized ova (9.4 +/- 1.3 vs 10.1 +/- 1.2 vs 7.5 +/- 1.1; P < 0.05) or transferable embryos (8.2 +/- 1.2 vs 8.4 +/- 1.3 vs 6.5 +/- 0.9; P < 0.05). Variation in the numbers of CL (P = 0.1) and unovulated follicles > or = 8 mm (P < 0.01) was lower in the ablation groups than in the steroid-treated group. Results suggest that follicle ablation is as effective as estradiol plus progesterone in synchronizing follicular wave emergence for superstimulation in cattle, and that ablation of the 2 largest follicles is as efficacious as ablating all follicles > or = 5 mm.     

Are the spectral Doppler indices of ovarian arteries indicative of antral follicular development and predictive of ovulatory responses and embryo yields in superovulated ewes?

Nineteen ewes received 200 mg of pFSH administered in eight decreasing doses from Days 1 to 4, starting three days before CIDR® device removal. Ten ewes received an injection of 350 μg of estradiol benzoate at CIDR® device insertion (Group E) and nine animals served as controls (Group C). B-mode and spectral Doppler ultrasonographic examinations were performed daily throughout superovulatory treatment to enumerate ovarian antral follicles and to determine ovarian blood flow indices, respectively. There were no differences (P > 0.05) in superovulatory responses between left and right ovaries/uterine horns or the two groups of animals. End-diastolic velocity (EDV) and mean velocity (Vm) values were greater (P < 0.05) on Days 1 and 2, and peak systolic velocity (SVp) was greater (P < 0.05) on Day 3 in Group C than in Group E. In Group E 15 correlations was recorded among indices (SVp, Vm, EDV, flow velocity integral-FVI, and pulsatility index-PI) and follicles numbers in different size classes on Days 1, 2 and 4, and seven correlations among indices (SVp, EDV, Vm, and vascular resistance index-RI) and superovulatory/embryo results (numbers of regressing corpora lutea, numbers/percentages of degenerated embryos and viability rates) on Days 1, 2 and 3. In Group C, there were three correlations among EDV and RI and medium-sized/large follicle numbers on Days 1 and 3, and five correlations among indices (EDV, RI and PI) and superovulatory/embryo results (numbers of luteinized unovulated follicles, degenerated embryos and unfertilized eggs) on Days 2 or 4. There was a lack of consistency in the velocimetric correlates of antral follicle numbers and superovulatory responses between the left and right side. Therefore, the usefulness of ovarian arterial indices to predict ovine superovulatory outcomes remains equivocal and requires further confirmatory studies.     

Ultrasonographic determination of ovarian follicular. Development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (+/- SEM) number of corpora lutea (CL) (6.2 +/- 1.5 vs 10.7 +/- 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 +/- 1.7 vs 8.4 +/- 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.     

The effect of immunization of heifers against alpha 1-26 inhibin conjugate on the superovulatory response to pFSH

A total of 121 heifers was blocked by time and diet and then randomly assigned, within block, to an inhibin-immunized (I) or a control (C) group. Immunized heifers (n = 61) received a primary immunization (Day 0) with 0.33 mg of an alpha 1-26 bovine inhibin fragment-human serum albumin (HSA) conjugate injected with non-ulcerative Freund's and DEAE-dextran adjuvants. Booster injections were given on Days 28 and 56. Control heifers (n = 60) received HSA and adjuvants. On Days 56 and 83 the ovaries of heifers were examined by ultrasound to determine the ovulation rate, and blood samples were collected for antibody titer determination. On Day 84, 61 heifers (C, n = 30; I, n = 31) received a total of 24 mg of porcine follicle stimulating hormone (pFSH), while 60 heifers (C, n = 30; I, n = 30) received 12 mg im pFSH, which was administered twice daily for 4 d in decreasing doses during the mid-luteal phase of the estrous cycle. Luteolysis was induced with prostaglandin F(2alpha) analog. The heifers were artificially inseminated and were slaughtered 7 d after estrus. Embryos were recovered and morphologically graded on a scale of 1 to 5 (1 = excellent; 5 = degenerated). Antibody titers (percentage binding at 1:125 serum dilution) differed (P < 0.01) between Group C and I heifers at Days 56 (0.1 vs 30%) and 83 (0.2 vs 37%), and 26% of Group I and 1% of Group C heifers (P < 0.01) had twin ovulations on Day 83. The mean number of embryos recovered was reduced (P = 0.02) in Group I heifers (8.9 +/- 1.2) compared with C heifers (12.1 +/- 1.1); however, the mean number of freezable embryos (Grades 1 and 2) was not affected (P = 0.61) by immunization, and there was no interaction with pFSH (P = 0.36). Ovulation rate as well as embryo yield and quality were not different (P > 0.10) between Group C and I heifers when 12 mg pFSH were administered; however, immunization decreased the superovulatory response to 24 mg of pFSH.     

The effects of once or twice daily injections of pFSH on superovulatory response in heifers

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.     

Superovulatory response in anestrous ewes is affected by the presence of a large follicle

Superovulatory response to conventional treatment with eCG (1200 IU) and progestagen sponges (MAP, n = 9; FGA, n = 9; or controls without sponge, n = 6) was studied in Corriedale anestrous ewes. The follicular population just before the administration of eCG and the total ovarian response (large anovulatory follicles plus normal CL and prematurely regressing CL) to treatment were determined after laparotomy. Pretreatment with progestagen did not modify the number or class of follicles greater than 1 mm observed on the ovarian surface at the time of eCG administration (19 +/- 2.2 follicles vs 19 +/- 2.9 follicles, for pooled progestagen-treated groups and control groups, respectively; mean +/- SEM) but significantly decreased the number of large anovulatory follicles (4.7 +/- 1.0 vs 10.2 +/- 2.6; P < or = 0.01) observed following treatment. Progestagen-treated animals were classified according to the presence (n = 13) or absence (n = 5) of a large follicle (LF: > or = 4 mm diameter) on the ovarian surface at the time of eCG treatment; a qualitatively better superovulatory response was observed in ewes without large follicle (large anovulatory follicles: 1.6 +/- 0.7 vs 5.8 +/- 1.3, P < or = 0.05; normal CL: 7.0 +/- 1.4 vs 3.8 +/- 1.0, P < or = 0.1; normal CL/total ovarian response: 78.7 +/- 10.1 % vs 34.9 +/- 8.2 %, P < or = 0.01; for ewes without LF and ewes with 1 to 2 LF respectively). No differences were observed in the individual ovulatory response when comparing ovaries ipsilateral or contralateral to LF in a same animal, indicating that the effect of LF on the superovulatory response would be fundamentally systemic. This work shows that, similar to what occurs in cows, the presence of a large follicle at the time of gonadotropin administration decreases the superovulatory response in anestrous ewes.     

Ovarian response and endocrine changes in buffalo superovulated at midluteal and late luteal stage of the estrous cycle: a preliminary report

A study was designed to determine whether superovulatory and endocrine responses in buffalo differ when gonadotropin treatment is initiated at midluteal and late luteal stages of the estrous cycle. Twenty-eight buffalo were randomized into 4 groups (A, B, C and D). Buffalo in Groups A and B (n = 8 each) were superovulated with Folltropin (total dose 25 mg) and Lutalyse. Treatments in Group A were initiated between Days 8 to 10 (midluteal group) and in Group B between Days 13 to 15 (late luteal group) of the estrous cycle. Buffalo in Groups C and D (n = 6 each) were not superovulated and served as controls. Blood samples from all groups of buffalo were collected daily for plasma progesterone and estradiol determinations. The number of corpora lutea (CL) and unovulated follicles was recorded (following per rectum palpations) 5 or 6 d post-estrus. Buffalo in Groups A and B exhibited estrus in larger proportions and earlier (49.33 +/- 3.82 h and 46.67 +/- 2.46 h, respectively) than the control Groups C and D (77.33 +/- 5.33 h and 78.0 +/- 3.83 h, respectively). Mean number of CL was higher in Group B (3.38 +/- 0.46) than in Group A (2.25 +/- 0.75), however,the difference was not significant (P > 0.05). Plasma progesterone concentrations on the day of treatment were higher in late luteal superovulated and control groups than in midluteal superovulated and control groups. In both Groups A and B progesterone levels were significantly related (r = 0.78,0.76; P < 0.05) to the number of CL palpated after the superovulatory estrus. Progesterone levels on the day of estimation of ovarian response were approximately 4 times higher in Groups A and B than in Groups C and D. Peak estradiol concentrations were approximately twice as high in superovulated groups as in control groups.     

Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors

The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi (Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH (n=24), which consisted of eight pFSH injections, or FSH/eCG (n=24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2mg estradiol benzoate and the insertion of an intravaginal progesterone-releasing device on Day 0. On Day 4, superstimulatory treatments were initiated and 100mg pFSH was divided into twice daily decreasing doses over a 4-day period. In the FSH/eCG group, the last two doses of pFSH were replaced by two doses of eCG (150 IU eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg PGF(2α) was injected i.m. The intravaginal progesterone-releasing device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg GnRH 18 h later. All females were artificially inseminated with frozen-thawed semen from the same bull 6 and 18 h after GnRH treatment. Seven days after GnRH treatment, embryos/ova were recovered and classified. Follicular superstimulatory (number of follicles ≥6mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means±standard error. The FSH/eCG group had higher superstimulatory (33.8±3.9 compared to 23.8±2.6 follicles; P=0.03) and SOV (16.8±2.9 compared to 10.8±2.1 CL; P=0.10) responses. Although the number of total ova/embryos was not different between groups (8.2±1.8 compared to 5.9±1.4 for FSH/eCG and FSH groups, respectively; P=0.25), the number (5.8±1.3 compared to 2.6±0.7; P=0.02) and percentage (75.6±5.7 compared to 53.2±9.7%; P=0.05) of transferable embryos was greater for the FSH/eCG females. Therefore, there was improvement in follicular superstimulatory and SOV responses and embryo quality in FSH/eCG-treated females.     

Effects of FSH commercial preparation and follicular status on follicular growth and superovulatory response in Spanish Merino ewes

Ovarian follicular development was characterized in 24 Spanish Merino ewes to study effects of the follicular status and the FSH commercial product used on follicular growth and subsequent superovulatory response. Estrus was synchronized using 40 mg fluorogestone acetate sponges. The superovulatory treatment consisted in 2 daily i.m. injections of FSH from 48 h before to 12 h after sponge removal. Sheep were assigned randomly to 2 groups treated with 6 decreasing doses (4, 4, 3, 3, 2, 2 mg) of FSH-P or with 6 doses of 1.25 mL of OVAGEN. Growth and regression of all follicles > or = 2 mm were observed by transrectal ultrasonography, and recorded daily from Day 6 before sponge insertion to the first FSH injection, and then twice daily until estrus was detected with vasectomized rams. Differences were detected in follicular development from the first FSH injection to detection of estrus (-48 to 36 h from sponge removal) between groups. Administration of FSH-P increased the appearance of new follicles with respect to OVAGEN (6.3 +/- 0.7 vs 4.8 +/- 0.4; P < 0.05), and the mean number of medium (4 to 5 mm) follicles (8.9 +/- 1.2 vs 6.6 +/- 0.9; P < 0.05). However, the mean number of follicles that regressed in size after sponge removal (5.9 +/- 0.4 vs 3.3 +/- 0.4) and the number of preovulatory sized follicles that did not ovulate (60 vs 42.4%) were also higher in FSH-P treated ewes (P < 0.05). So, finally, there were no differences in ovulation rate, as determined by laparoscopy on Day 7 after sponge removal, between ewes treated with FSH-P or OVAGEN (6.3 +/- 1.9 vs 7.0 +/- 1.7 CL). In all the ewes, the ovulatory response was related (P < 0.05) both to the number of small follicles (2 to 3 mm in diameter) present in the ovaries at the start of treatment with exogenous FSH and to the number of follicles that reached > or = 4 mm in size at estrus, despite differences in the pattern of follicular development when using different commercial products.     

Effects of estradiol-17β and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet

Twenty-four cycling swamp buffaloes with normal reproductive histories and 2–3 months postpartum were used to investigate the effect of addition of estradiol-17β and human chorionic gonadotrophin (hCG) to the superovulation regime on the level of ovarian stimulation and embryo production.The estrous cycles of buffaloes were synchronized by prostaglandin injection and then divided into two groups for superovulatory treatment. Those in Group 1 (n = 12) received a implant containing 3 mg norgestomet (Syncro-Mate-B) for 9 days (insertion day is Day 0), with 4000 IU of equine chorionic gonadotrophin (eCG) and 500 μg cloprostenol i.m. given at Day 7. Group 2 (n = 12) received the same regime as Group 1, together with 7.5 mg estradiol-17β given in three intramuscular injections on Days 3, 5 and 7 in decreasing doses (4.0, 2.5 and 1.0 mg, respectively) and 5000 I.U hCG i.v. coincidentally with the first insemination. Estrus was monitored visually and by placing treated animals with bulls. Each animal was inseminated twice with frozen sperm after standing estrus. The numbers of corpora lutea (CL) and follicles greater than 8 mm in diameter were recorded via palpation per rectum at 6 days after implant removal. Two days later 11 animals from Group 2 and two from Group 1 were slaughtered for direct observation of ovarian responses and for embryo collection.The mean number of CL were 0.91 ± 0.66 and 9.08 ± 5.0 for Groups 1 and 2, respectively. The average recovery rate based on CL counts at slaughter was 60% in Group 2. No embryos were recovered from the two animals in Group 1. Seventy-nine percent of the collected ova were fertilized and more than 60% of them had developed into hatched blastocysts. The percentages of buffalo with excellent and good estrus were 41.6 and 91.6% for Groups 1 and 2, respectively.These results showed that the supplementation of estradiol-17β and the hCG treatment significantly improved the level of ovarian stimulation in swamp buffalo.     

Effect of estradiol benzoate or GnRH treatment prior to superstimulation in CIDR-treated, Korean native cows (Bos taurus)

The objective of this study was to evaluate the effectiveness of superovulatory protocols by synchronizing the emergence of the follicular wave using estradiol benzoate (EB) or GnRH in CIDR-treated, Korean cows. Sixty-six cows were used in the study and these were divided into three groups. The standard group comprised cows that were between days 8 and 12 of their estrous cycle (n=22). The remaining 44 cows, at all other stages of the estrous cycle, received CIDR and were assigned to two treatment groups that received either 2mg EB (EB-CIDR group, n=22) or 100 microg GnRH (GnRH-CIDR group, n=22) 1 day after CIDR insertion. Gonadotropin treatment began between the 8th and 12th days of the estrous cycle in the standard group, 5 days after EB injection in the EB-CIDR group, and 3 days after GnRH injection in the GnRH-CIDR group. All cows were superovulated with porcine FSH (pFSH) twice daily, with the dose (total 28 mg) decreasing gradually over 4 days. On the 5th and 6th injections of pFSH, 25 and 15 mg doses of PGF(2alpha) were administered. CIDR was withdrawn at the 7th pFSH injection and the cows received 200 microg GnRH at 24h after CIDR withdrawal. Cows were artificially inseminated twice at 36 and 48 h post-CIDR withdrawal and embryos were recovered 7 days after the 1st insemination. The numbers of preovulatory follicles (22.9-28.2), ovulated preovulatory follicles (17.6-21.7) and CL (15.9-17.9) detected by ultrasonography did not differ among groups (P>0.05). Similarly, the numbers of total ova (6.7-10.0), transferable embryos (4.0-6.0), degenerate embryos (1.1-1.8) and unfertilized ova (1.3-4.3) did not differ among groups (P>0.05). Progesterone and estradiol concentrations during superovulation treatments and at embryo recovery were also the same in all groups (P>0.05). We conclude that in CIDR-treated Korean native cows, superovulatory treatments that follow administration of either EB or GnRH (at any stage of the estrous cycle) result in both a superovulatory response and embryo yield comparable to conventional superovulation protocols.     

Effect of follicular status on superovulatory response in ewes is influenced by presence of corpus luteum at first FSH dose

The present study was developed to assess possible effects on ovulatory response and embryo yields arising from the presence of a corpus luteum (CL) at the time of initiation of the progestagen treatment used in superovulatory protocols in sheep. In breeding season, estrus was synchronized in 25 Manchega ewes using 40 mg FGA sponges for 14 days, together with a single dose of 125 microg of cloprostenol on Day 12, with Day 0 as day of progestagen insertion. Superovulatory treatment consisted of eight decreasing doses (1.5 x 3 ml, 1.25 x 2 ml, and 1 x 3 ml) of Ovagen twice daily from 60 h before to 24 h after sponge removal. The presence or absence of corpora lutea was assessed by transrectal ultrasonography at progestagen insertion and at first FSH dose. Number and size of all follicles > or = 2 mm were also evaluated at first FSH dose. The number of corpora lutea and the number and viability of recovered embryos in response to the treatment were evaluated 7 days after sponge removal. No significant effect on ovarian response of the presence of a CL at sponge insertion in 21 of the 25 ewes (84%) was detected. However, ewes with a CL at first FSH dose (16 ewes, 64%) yielded a higher number of transferable embryos (7.2 +/- 1.4 versus 2.7 +/- 0.7, P < 0.05), since the embryo degeneration rate was increased in sheep without a CL (42.5% versus 12.7%, P < 0.01). Analysis of possible effects derived from the presence of a large presumptively dominant follicle (> or = 6 mm) at first FSH dose showed that both recovery and viability rates were lowest (P < 0.05) in ewes bearing a large follicle in the absence of a CL (40.5 and 50.6%, respectively), and highest in ewes that did not show a large follicle but in which a CL was present (73.9 and 85.2%). The final number of transferable embryos was very different between groups (10.2 versus 1.8, P < 0.01). These results indicate that the number and quality of embryos obtained from superovulated ewes is affected by the presence of a CL prior to the first FSH dose (i.e. by the stage of the estrous cycle at progestagen insertion) and also by an interaction with suppressive effects from large dominant follicles. This finding suggests the existence of some effects on follicular population prior to the FSH treatment that may compromise follicle and oocyte developmental competence. It seems reasonable to hypothesize that superovulatory yields would be increased by beginning the treatment during the early-luteal phase of the estrous cycle, allowing for the presence of a CL along with the progestagen treatment.     

The effects of dose and duration of administration of pFSH during the first follicular wave on the ovulation rate of beef heifers

Four experiments were carried out to examine the effects of administration of pFSH (Vetrepharm) from Day 3 of the estrous cycle in conjunction with PG on Day 5 on follicular populations and ovulation rate in heifers. In Experiment 1, 47 heifers were allocated to 1 of 4 treatment groups (n = 11 to 12 per group): a) control, b) 1.5 mg pFSH, c) 2.0 mg pFSH or d) 2.5 mg pFSH until estrus. Heifers assigned to the 3 treatments had a higher ovulation rate than the controls (P < 0.05). In Experiment 2, 45 heifers were allocated to 1 of 5 treatment groups (n = 8 to 10 per group): a) control, b) 1.0 mg pFSH until PG, c) 1.0 mg pFSH until estrus, d) 1.5 mg pFSH until PG or e) 1.5 mg pFSH until estrus. From Day 5, heifers assigned to pFSH treatments had more large follicles than the controls (P < 0.05). There was no effect of treatment on the incidence of twin ovulations. In Experiment 3, 43 heifers were assigned to 1 of 3 groups (n = 11 to 16 per group): a) control, b) 1.0 mg pFSH until estrus or c) 1.5 mg pFSH until estrus. At slaughter, 14 d after administration of PG, the incidence of twin ovulations was 0/11, 7/16 and 8/16 for Groups a, b and c, respectively (P = 0.011). In Experiment 4, pFSH (1.5 mg) was administered to 3 groups during the development of the first dominant follicle: a) growth phase (n = 19); b) static phase (n = 17); and c) decline phase (n = 17). All pFSH-treated heifers had a higher ovulation rate than the controls (P < 0.05); heifers assigned to Group c had a higher ovulation rate than those in Groups a or b (P < 0.05). More heifers assigned to Group c (7/17) superovulated than in the other 2 groups (P < 0.05). In conclusion, administration of 1.0 or 1.5 mg pFSH twice daily beginning at Day 3 of the estrous cycle in association with the induction of luteolysis increased the ovulation rate significantly when pFSH treatment was continued to onset of estrus. The ovulation rate and the occurrence of multiple ovulations were significantly higher when pFSH was administered at the time that the first dominant follicle was in decline.